Nucleotide linked to protein through phosphodiester bonds has not been found in normal eucaryotic cells. A sensitive detection methodology for this post-translational modification may be possible by the measurement of tyrosine phosphate which is produced by micrococcal nuclease treatment of nucleotidylylated protein. Quantification of tyrosine phosphate before and after enzymatic treatment can be used to detect phosphodiester linkage of nucleotide to protein. Studies on the ability of a nuclease to remove adenosine from adenylylated glutamine synthetase found that the native protein is resistant to nuclease attack while the denatured form readily undergoes deadonosylation. This may indicate that posttranslational modification sites of proteins are protected from nonspecific enzymatic attack by tertiary or quaternary structural factors which may be involved in the control of enzymatic interconversion of proteins. Nucleotide specificity requirements were studied for the adenylylation of glutamine synthetase with [Alpha-32P]ATP [Alpha-32P]AMPPNP and [Alpha-35S] thiophosphate derinatives of ATP. The Alpha-thiophosphorylated ATP but not AMPPNP was utilized as a substrate. It was not possible with the deadenylylating enzyme to cleave the thiophosphodiester bond to glutamine synthetase. Snake venom phosphodiesterase binds adenylylated glutamine synthetase about 1000-fold tighter than a small molecular weight substrate, nitrophenyl dTMP(5'). The tighter binding is inversly correlated with catalysis.